A mechanical transition from tension to buckling underlies the jigsaw puzzle shape morphogenesis of histoblasts in the Drosophila epidermis

The polygonal shape of cells in proliferating epithelia is a result of the tensile forces of the cytoskeletal cortex and packing geometry set by the cell cycle. In the larval Drosophila epidermis, two cell populations, histoblasts and larval epithelial cells, compete for space as they grow on a limited body surface. They do so in the absence of cell divisions. We report a striking morphological transition of histoblasts during larval development, where they change from a tensed network configuration with straight cell outlines at the level of adherens junctions to a highly folded morphology. The apical surface of histoblasts shrinks while their growing adherens junctions fold, forming deep lobules. Volume increase of growing histoblasts is accommodated basally, compensating for the shrinking apical area. The folded geometry of apical junctions resembles elastic buckling, and we show that the imbalance between the shrinkage of the apical domain of histoblasts and the continuous growth of junctions triggers buckling. Our model is supported by laser dissections and optical tweezer experiments together with computer simulations. Our analysis pinpoints the ability of histoblasts to store mechanical energy to a much greater extent than most other epithelial cell types investigated so far, while retaining the ability to dissipate stress on the hours time scale. Finally, we propose a possible mechanism for size regulation of histoblast apical size through the lateral pressure of the epidermis, driven by the growth of cells on a limited surface. Buckling effectively compacts histoblasts at their apical plane and may serve to avoid physical harm to these adult epidermis precursors during larval life. Our work indicates that in growing nondividing cells, compressive forces, instead of tension, may drive cell morphology.

The color code was initially in the caption.Indeed, the reviewer is right that it is also needed in the figure panel.We thus added it.
Lines 195-208.The authors refer to Fig. 8E-H; Fig. 5 would probably be correct.We thank the reviewer for pointing out the mistake, which has been corrected.We added this information in the figure caption, cad::mkate was used to mark cell junctions.We also have indicated all genotypes of the study in table 2. Fig. 7C.The authors show that increased LEC growth (TSC-RNAi) results in reduced histoblast area.To further test their hypothesis, the authors should determine whether in this situation also the buckling of cell junctions (circularity) is affected (like the authors do for the reduced LEC growth (Fig. S5)).This is a challenging question.There is indeed an effect on lobule shape, narrower and more packed, as visible in Fig 7 .This effect verified by the quantification of the junction straightness (defined by the ratio of end-to-end Euclidean distance over the curvilinear distance along the junctions), which decreases from 0.67 (WT, median value) to 0.57 (tsc1-RNAi, p = 0.004).However, we do not measure a significant change in the circularity value.We believe this is due to two main reasons.First, the calculation of circularity depends on the precise measurements of cell area and perimeter.But in e22c>tsc1-RNAi TSC1-RNAi larvae, the nests are so shrunk that our segmentation fails, and individual cells are not detected correctly.Quite frequently, the opposite cell membranes even come into contact, transforming one convolved cell into several less convolved rounder cells (see figure below).Note that the straightness is less sensitive to this because a full segmentation of the cell is not required (segmented junctions or pieces of junctions are enough).We have added the statistics on straightness index lines 300.See below how one orange cell with low circularity can be transformed by segmentation error into 3 cells, 2 with high circularity and one with intermediate.This messes up the statistics.We haven't found an easy way (area cutoff) to remove these artifacts.
Second, we believe that there is also a non-autonomous effect on the growth of histoblasts' junctions.In fact, we observed decreased cell perimeter, that cannot be explained only by the artifact mentioned above.This could be expected, as a compressed cell is likely to yield contact inhibition-type feedback, complex to analyze.Nevertheless, the experiment does provide useful information.In particular, it adds evidence of the tug-of-war mechanism that controls the apical nest size and, consequently, the junction shape.************ Rev. 2: As I stated in my first review, the observation of histoblast junctional buckling is very interesting.Also, the authors' hypothesis, presented at the end of the discussion, that histoblasts buckle to ensure that they are not accidentally pushed out of the tissue/die during larval growth is intriguing.
In this second revision, we present new experiments which strengthen our mechanical description of histoblast.We confirm that indeed histoblasts are in a mechanical state different from the one of embryonic tissues, for example.We think the work will interest a wide audience working on tissue mechanics in a developmental context.
Overall, the authors have sought to address all my comments and have improved the manuscript.As part of the revision, the authors have adapted their line of argument.Based on their data, the authors formulate the hypothesis that junctional lengthening combined with a reduction in histoblast area leads to junctional buckling.For some reasons histoblasts can't withstand this pressure like LECs.Indeed, as growth proceeds on the epidermis (LECs and histoblast), pressure builds up.This pressure varies smoothly over the surface of the epidermis and both histoblasts and LECs probably experience it.As we argue in the discussion lines 342-348, there are several possible factors that make histoblasts more compliant, and more prone to buckling of their junctions than LECs.
Although the revision has improved the manuscript, two main points remain, which in my opinion need to be addressed further before the manuscript can be published in PLoS Biology.We thank the reviewer for stressing some potential misunderstandings regarding the mechanics of histoblasts.This pushed us to clarify our statements and make important additional experiments.Some of them (optical tweezers on living larva) were a real technical challenge.But they helped us to strengthen our point.See below.
1) The manuscript does not sufficiently explore the mechanism behind the behavior of the buckling junctions.See below for our specific answer on histoblast junction mechanics: we now provide evidence that the mechanics of junctions are dominated by elasticity.As they grow in a constrained domain, they buckle.
How are the forces created that lead to junctional lengthening, and what stabilizes the buckled junctions?Similar to other epithelial systems, a major driving force is the accumulation of junctional adhesive proteins in these growing cells.Our experiments show that altering trafficking of the endocytic pool (rab11-DN) or reducing the total production of cadherins (Cad-RNAi) results in smaller junctions and no buckling.This is a crucial point, and our previous manuscript did not thoroughly explore the cadRNAi experiment.Consequently, in this revised manuscript, we present new cad RNAi experiments in which we observe the apical side of histoblasts with a membrane marker.The phenotype is impressive, and we observed no lobulations in all the wandering stage larvae that we analyzed.An image has been added to Fig. 3D,E, and the quantifications are included in Fig. 4G-I.In growing dividing cells, junctional material increases, but junctional growth is balanced by cell divisions, limiting the growth of an individual junction to the cell cycle duration.However, when we consider the integral of junctional length at the cell population level, it does show growth.In these other systems, no buckling occurs because 1) junctions are kept short by continuous cell divisions, as discussed in the cdc25 experiments (lines 164: 'Would the histoblasts divide like, for example, cells of the imaginal discs, the deep junctional lobules would not occur'); 2) these systems are usually not under compression.'What stabilizes the buckled junctions?' : In an inert elastic system, one would expect the buckled shape to persist as long as compressive stress is maintained.However, in cells, quasi-static processes associated with the renewal of junctional components and growth come into play.This is what we address in the ablation experiments, which we refer to as the plastic process (see below).
Laser ablation experiments show that there is hardly any relaxation when junctions of buckled histoblasts are cut, which the authors interpret as a 'plastic process [that] dissipates the compressive stress'.What is this process?We observe no relaxation upon ablation, thus the stress that generated the deformation has dissipated.Plastic processes are non-reversible changes of shape in response to applied forces.Because junctions undergo important deformations, it is plausible that the dissipative process at play in junctions is of plastic nature.However, we have no formal proof and following the reviewer's comment, we have used predominantly "a dissipative process" in the revised manuscript.We have added (line 230) a comment saying that as of now, we do not know if the stress dissipation is triggered by deformation (plasticity), or if it is time that matters (viscosity).Note that we demonstrate with new experiments (see below) that on a short time scale, histoblasts are elastic (no dissipation).Thus, our current mechanical interpretation of late larval histoblasts is that of an elastic structure, which dissipates only at a very long time/ deformation.Most importantly, in our work, we show in the simulation that this is compatible with the existence of buckling.Regarding the molecular nature of the dissipative process, we speculate in the simulation section (line 229) and in the myosin II section (line 273) that it could stem from reorganization of the cytoskeleton.
In addition, the model includes 'an elastic fabric impeding the rapid displacement of the boundary'.What is this 'elastic fabric'?see below for a detailed answer.Note that we have decided to change the terminology for the more canonical expression (in mechanics terminology) "elastic foundation".Do these observations suggest that the junctions, although they are strongly bent, are very stiff/rigid?Would this depend on a rigid cortical actomyosin network?We do think indeed that they are elastic structures (in the sense that they are not as dissipative as embryonic junctions).This is a central point that we did not explained clearly enough in the previous manuscript-and we thank the reviewer for pointing this out.As suggested by the reviewer (below), we have added analysis from live imaging to show that the tissue is really static compared -for example-to embryonic tissues.This corresponds to Fig S3 .This figure is first mentioned in lines 73-79 in the text.We then refer to it again line 189.In this revised manuscript, we investigate more thoroughly the mechanics of histoblasts (paragraph starting line 188) by adding a critical experiment : optical tweezers that show that when you stimulate a point on a junction, the entire region moves in synchrony.The overall structure is elastically interconnected.This corresponds to Fig 4E-H, Movie S1 and lines 190-207 in the text.By comparison, the same experiment in embryos does not display such an interconnection : the mechanical oscillation does not propagate to neighboring regions (this has been reported previously in Clement et al. cited in our manuscript and we reiterate the measurement by sake of completion in fig S5, and Movie S2).Moreover, we show in the tweezer experiment that different junctions seem to be interconnected through the cell medium (cytoplasm or apical cortex).This elastic connection serves the role of an "elastic foundation" (a classic mechanics terminology) that prevents large, low-mode deflection of the beam.In the previous manuscript we had used the term "elastic fabric" thinking this could be more intuitive when talking about a tissue.Clearly, it was a mistake and we now stick to "elastic foundation".While indeed this very likely depends on an elastic cortex, we stress that the whole apical domain of histoblast may become elastic through its connection to the stiff apical extracellular matrix (mentioned line 343).Note that the junctions are elastic on short time scales, but on longer times scales they adapt to their bent morphology (what we call the dissipative process).As we mention in the discussion (line 339), this is reasonable that on the time scale at which growth operates, there is some renewal of junctional components that could help release mechanical stress.Overall, through the added experiments, and the reformulation of our interpretation of experiments, we think the revised manuscript is clearer on the description of histoblast mechanics -making the results stronger and all the more original.
Here, the actin and myosin data seem counterintuitive.This highlights that the role of the described changes in actomyosin is not sufficiently clear.First, and as we mentioned in the previous version of the manuscript, we have toned down our statements with respect to the cytoskeleton since our first version of the manuscript.But we want to mention that 1) while we state that junctions in histoblast are elastic rather than dissipative (except at very long times scales), a reduction in stiffness can still happen, which would promote buckling through a reduction of the critical buckling load, rather than impede.2) An enrichment in actomyosin in the apical medial medium could increase the stiffness of this region and promote elastic coupling of junction to the elastic foundation.These elements are discussed in lines 264-270 in the results section and 348-349 in the discussion.
To explore the properties of the buckling junctions in more detail, live imaging would be useful.From the authors' data, one would expect that the buckled junctions are very stable and do not change much over time (in contrast to dynamic changes in the buckling morphology, which one would expect if one would deform a flexible, already buckled rod).
Such live imaging would also help to illustrate the statement made in line 74: 'No fluctuations of the cell junctions are observed in a period of minutes.'.As detailed above, following the reviewer's comments, we have performed live imaging.The junctions are indeed static --or rather quasi-static as there are still some slow dynamics (building-up of lobules as the junctions grow).We also have performed optical tweezers experiments which bring new assessment of our hypothesis.It stands that when mechanically stimulated at one point, the whole surrounding moves in a quite coherent fashion (see movie S1).We thus fully agree with the reviewer in their above statement.The corresponding changes in the manuscript text and Fig 4 have been detailed above.
2) The manuscript still does not provide sufficient support for the hypothesis that pressure from the LECs is causing histoblast buckling.One experiment that could expand on the InR-DN and TSC1-RNAi experiments would be to ablate one LEC neighboring the histoblast nest to release tension and ask if and how junctional morphology of the histoblasts changes.While this is a very interesting suggestion, we think that such an experiment is likely to generate active processes of wound healing and compensatory growth that would make it hard to interpret.
For the InR-DN and TSC1-RNAi experiments, it would be helpful, if the authors explained in more detail which LECs were assessed for their cell area (all of them, those neighboring the histoblasts?).We measured the area of LECs within 3 cell diameters of histoblast nests.We measured LECs area on the same larva that were used for histoblast measurements -preventing variability associated with fitness and precise developmental stage.Owing to the fact that the same buckling occurs in dorsal, ventral, anterior, posterior nests of all segments, we assume that there is no regionalization of LECs growth either.Most importantly, for the perturbative experiments, e22c-Gal4, which is an epidermis driver used in many studies, drives expression in all LECs, in an unregionalized fashion (see below, last question).
If all LECs of the abdomen got bigger or smaller, would this not have strong effects on epithelial morphology, as a large area is either created or lost (or are the larvae smaller/larger)?Are LEC numbers in one segment similar in InR-DN, TSC1-RNAi and control experiments?The change in LECs is not immense.This is at least partially buffered by compression of histoblast nests.Note that because LECs cover a large portion of the epidermis, a small change in LECs can mean a big change for histoblasts.Note that there is no cell divisions in LECs in the developmental stages that we analyse.Thus there is no change in cell number in these experiments.Also, it would be useful to know not just the cell area of individual histoblasts, but also the overall area of the nests in these experiments.This would indicate the area available for the histoblasts and further highlight the change in available space for the histoblasts.(This would also be interesting for the wild-type analysis in Fig. 1).Following the reviewer's comment, we also include the change in area at the nest scale, lines 303-307, where it is indeed quite relevant.Also, does junctional tension change in inR-DN and TSC1-RNAi LECs?Could a detailed study of the morphology of LECs and histoblasts by live imaging at the start of buckling help to explore whether LEC pushing is involved?For instance, does buckling begin in certain areas of the nest, which maybe correlate with specific LECs getting larger?Also, the authors discuss that buckling begins later at the LEC/histoblast interface -is this because of more rigid junctional tension or because LECs actively push?We do not say in this or the previous version of the manuscript that LECs are pushing.Throughout the manuscript, we talk about pressure build up, as cells grow, instead of "LECs pushing".Our view is not of LECs performing an active process against histoblast at the LECs-histoblast interface.Although an interesting idea, we haven't seen anything like this.Rather, as growth proceeds on a limited surface, pressure builds up --ie compression.In a way, histoblast could contribute as well to this pressure build up.But they occupy a much smaller surface, and grow slower.To prevent any possible misunderstanding, we strived to use the proper terms in the revised manuscript.Straight from the first introduction of the buckling hypothesis, we make it clear that it is a matter of a balance between growth of junctions and growth/shrinkage of the apical domain of histoblast (line 127).We repeat this at several other instances -for example when we introduce simulations (line 209-213), where we stress that potentially, buckling could proceed with an apical domain that is stationary or even grows -if this growth is slow enough that it does not balance the fast growth of junctions.In response to the reviewer's comment on the location of buckling, there is something very interesting that we're exploring currently --not about position but orientation.There is an asymmetry in the change in nest shape: it shrinks more along the antero-posterior axis.and on average the junctions that buckle first are oriented along the antero-posterior axis.This is something we're currently exploring, but remains for a future study.

Fig. 3E .
Fig. 3E.The authors should state in the legend what the color code refers to.The color code was initially in the caption.Indeed, the reviewer is right that it is also needed in the figure panel.We thus added it.

Fig. 7C .
Fig. 7C.The authors should state which marker (cad...) they have used.We added this information in the figure caption, cad::mkate was used to mark cell junctions.We also have indicated all genotypes of the study in table 2.